Scanning electron microscopy of Onchocerca fasciata (Filarioidea: Onchocercidae) adults, microfilariae and eggs with notes on histopathological findings in camels.

BACKGROUND: Onchocerca fasciata is a prevalent filarial species in camelids of Asia and Africa forming nodules in the skin of dromedary and Bactrian camels. In spite of recent advances in the biology and epidemiology of this nematode species, a relatively scant number of studies have focussed on the morphology of this parasite. The main objective of this study was to describe morphological characteristics of adults, microfilariae and eggs of O. fasciata by scanning electron microscopy (SEM), staining and histology. METHODS: From April 2016 to March 2017 dromedary camels (n = 456) were inspected for infection with O. fasciata in a slaughterhouse in Kerman (south of Iran). Adult worms in nodules were isolated by digestion of nodules in collagenase and used for SEM. Skin nodules were also fixed, sectioned and stained with hematoxylin and eosin for histopathology. Skin microfilariae that were isolated from tissues surrounding the nodules were confirmed as O. fasciata by sequencing of the cytochrome c oxidase subunit 1 (cox1) and 12S rRNA genes and used for SEM and Giemsa staining. RESULTS: Single or multiple O. fasciata nodules (1.2-2.2 cm in diameter and 507-845 mg in weight) were found in 30.3% of the examined camels. SEM analysis helped identify 18 papillae in the caudal region of the male. Discontinuous longitudinal cuticular crests were observed in the posterior region of the male. In female nematodes, the ridges had a rounded shape with a height/width ratio of 7/16 in longitudinal sections. Unsheathed skin microfilariae with a rounded anterior extremity measured 210.7 × 2.5 µm on average. Developed eggs containing microfilariae measured 35.9 × 31.0 µm and their smooth shell surface had characteristic tongue-like appendages. In addition to inflammatory reactions surrounding the parasites, accumulation of intracellular ceroid pigment, golden-yellow to brown in colour, was observed within macrophages upon histopathological examination. CONCLUSIONS: We found longitudinal crests on the surface of the posterior region of the male nematode. Measurements of the main morphological features of microfilariae and eggs, and the shape index of ridges (height/width) in female nematodes are described for the first time.

Morphological and molecular characterization of Onchocerca fasciata (Nematoda, Onchocercidae) from dromedary camels (Camelus dromedarius) in Iran.

Skin nodules of Onchocerca fasciata Railliet and Henry, 1910 (Spirurida, Onchocercidae) are a common finding in dromedary camels, though with a minimal clinical impact. There is little information about the morphology, molecular make-up and pathological impact of this parasite. Onchocerca fasciata nodules (1.3-2.1 cm in diameter and 509-841 mg in weight) were detected on the neck region in 31.5% of dromedary camels examined in Kerman province, southeastern Iran. Of 38 isolated nodules, only 23 (60.5%) contained viable worms. Measurement and morphological analyses were performed on isolated female worms by light microscopy. The identification of O. fasciata specimens was confirmed by sequence analysis of two mitochondrial genes (12S rDNA and cox1), which showed 0.4% divergence from available O. fasciata sequences. In addition, a phylogeny of filarial nematodes was constructed, based on these two mitochondrial genes and five nuclear genes (18S rDNA, 28S rDNA, MyoHC, rbp1, hsp70); this indicated that O. fasciata belongs to clade ONC3 of Onchocercidae, with representatives of the genera Onchocerca and Dirofilaria. Within the genus Onchocerca, O. fasciata is grouped with bovine parasitic species and the human parasitic Onchocerca volvulus, which suggests an impact of domestication on the radiation of the genus. Data provided here on the distribution and morphology of O. fasciata contribute to the molecular identification and phylogenetic position of the species.

Ocular onchocercosis in dogs: aberrant infection in an accidental host or lupi onchocercosis?

Four adult dogs that had spent their entire life in Hungary, were found to be infected with filaroid nematodes of the genus Onchocerca. The morphology and location of the parasites as well as pathological lesions were similar to those described earlier in the one Hungarian and five US dogs. Only moderate morphological differences were noted between the adults of Onchocerca sp. infecting dogs and O. volvulus of man or O. lienalis of cattle. Nevertheless, the morphology of microfilariae of Onchocerca from dogs is unique within the genus. Their length was less than half the length of microfilariae of other Onchocerca spp. known so far. In addition to size differences, several characteristic morphological features were observed. The unsuccessful attempt to infect dogs with O. lienalis, the absence of O. volvulus and O. lienalis in endemic regions of canine onchocercosis, the different size, morphology, and location of the adults in dogs and cattle, the exceptionally small size and unique morphology of microfilariae of Onchocerca of canids indicate that a distinct species might be responsible for canine onchocercosis. Since the larval concentration in the skin was high (50-3600 microfilariae g(-1)) in all affected dogs, the diagnosis prior to surgical removal of worm nodules can be based on the examination of a small skin snip collected from the head or abdominal region. Infections in dogs may provide a model to study human onchocercosis, therefore, further studies are encouraged on the feasibility of experimental infection of dogs with this Onchocerca species.

Canine ocular onchocercosis in Hungary.

An adult male mongrel dog that had spent its entire life in Hungary, was found to have infection with filaroid nematodes of the genus Onchocerca. The gravid male and female parasites were embedded in bean-sized granulomatous masses on the conjunctiva and the sclera of both eyes. The cuticle of females consisted of two separated layers in longitudinal sections, the external layer bearing ridges and the internal layer showing striations. The ridges were marked, rounded in shape, and the ratio of body diameter to the distance between ridges varied between 7:1 and 10:1. At midbody of the worms, two striations could be seen between each pair of ridges: one under every ridge and one between neighbouring ridges. Numerous exceptionally small (96.4 microm x 6.4 microm) microfilariae were seen in the uteri of females and the surrounding tissues and isolated from skin biopsy materials. The morphology and location of the parasite and histopathological lesions of the Hungarian case were similar to that described in dogs in the United States. This case is the first documented ocular Onchocerca infection in dogs outside the western United States. Thus, onchocercosis should be considered in the differential diagnosis of ocular and periocular nodules in dogs also in Europe.

In-vitro uptake of ivermectin by adult male Onchocerca ochengi.

Ivermectin is not lethal to the adult worms of Onchocerca volvulus or to those of O. ochengi, a cattle parasite closely related to O. volvulus. Although ivermectin penetrates the nodules in which the adults of these nematodes live, it is not known what levels of the drug enter the worms. Adult male O. ochengi were incubated in [3H]ivermectin in a saturated solution of unlabelled ivermectin (11.44 microM), to measure uptake by the oral and transcuticular routes, and in [3H]inulin, to ascertain if oral ingestion occurs in vitro. Uptake of [3H]ivermectin was high [1040 disintegrations/min (d.p.m.) at 3 h, representing a mean total of 86 pmoles ivermectin/worm] and occurred predominantly by the transcuticular route. Viability of worms was not reduced by this exposure, and uptake continued for up to 12 h. Only low levels of [3H]inulin (four d.p.m.) were detected in worms, indicating that the gut is probably not functional in vitro. Scanning and transmission electron microscopy revealed that the epicuticle of both sexes had an irregular surface which was pitted with a honeycomb structure in males, and rough and abundantly folded in females. These structures greatly increased the absorptive surface of the worms. In conclusion, ivermectin is able to enter adult O. ochengi males at concentrations sufficient to kill non-filarial nematodes.

Electron microscope observations on Onchocerca ochengi and O. fasciata (Nematoda:Filarioidea).

The fine structure of the females and males of Onchocerca ochengi (parasitizing zebu and cattle) and of the females of O. fasciata from camels were described and compared to other filariae of the genus Onchocerca. It was shown that O. ochengi resembles O. volvulus of humans in its degree of development, while being more primitive than O. gibsoni. Besides other similarities O. ochengi attracts inflammatory cells in the way of O. volvulus and these could be a model for chemotherapeutic trials.

Identification of a common filarial larva in Simulium damnosum s.l. (type D, Duke, 1967) as Onchocerca ramachandrini from the wart hog.

Filarial larvae resembling Type D (Duke, 1967), which are common in the Simulium damnosum s.i. vectors of human onchocerciasis ("riverblindness") in several parts of West Africa, were dissected from wild-caught flies in north Cameroon and examined morphologically. This was done in order to establish a possible synonymy with infective larvae (L3) of 2 recently discovered Onchocerca species of wart hogs (Onchocerca ramachandrini Bain, Wahl, and Renz, 1993 and Onchocerca sp. Wahl and Bain, 1995), which had been found to resemble Type D. After dissection of approximately 1,700 S. damnosum s.1., 13 Type D-like larvae were recovered from 12 infected flies. Their morphology corresponded to O. ramachandrini.

Onchocerca fasciata: enzyme histochemistry and tissue distribution of various dehydrogenases in the adult female worm.

Histochemistry studies of key dehydrogenases in the glycolytic pathway and related enzymes and the tricarboxylic acid (TCA)-cycle enzymes were carried out on adult female Onchocerca fasciata. The distribution pattern and enzymatic activities of 6-phosphogluconate dehydrogenase (6-GPDH), lactate dehydrogenase (LDH), mitochondrial glycerol-3-phosphate dehydrogenase (GPDH), nicotinamide adenine dinucleotide (phosphate) [NAD+(P)]-linked isocitrate dehydrogenase (ICDH), and NAD+(P)-linked malate dehydrogenase (MDH) in various tissues of the worm were determined. Moderate to intense enzyme activities were localized in three main areas, namely, the hypodermis, body-wall muscle, and reproductive tissues. Activity of the formazan reaction product was very low, if at all present, in the intestinal epithelium and was completely absent in the cuticle. On the basis of the present results and earlier observations, it is suggested that glycolysis leading to the end product lactate is the main energy-generating pathway in O. fasciata. The presence of significant activity of 6-GPDH indicates that the pentose-phosphate pathway might be operative in O. fasciata. In light of the activity of some of the TCA-cycle enzymes, ICDH and MDH, demonstrable in O. fasciata, it is possible that an additional pathway (pyruvate-succinate) of glucose metabolism via a reverse sequence of the TCA cycle may also be operative in the worm. The possible functional significance of the enzymes detected is discussed with respect to their location.

Molecular cloning and characterization of onchocystatin, a cysteine proteinase inhibitor of Onchocerca volvulus.

A cDNA clone designated OV7 encodes a polypeptide that corresponds to a highly antigenic Onchocerca volvulus protein. OV7 has significant amino acid sequence homology to the cystatin superfamily of cysteine proteinase inhibitors. In this report we establish that the OV7 recombinant protein is active as a cysteine proteinase inhibitor, and we have named it onchocystatin. It contains a cystatin-like domain that inhibits the activity of cysteine proteinases at physiological concentrations. Recombinant glutathione S-transferase-OV7 (GST-OV7, 1 microM) and maltose-binding protein-OV7 (MBP-OV7, 4 microM) fusion polypeptides inhibit 50% of the enzymatic activity of the bovine cysteine proteinase cathepsin B. Neither fusion polypeptide inhibits serine or metalloproteinases activity. The Ki for GST-OV7 fusion polypeptide is 170 nM for cathepsin B and 70 pM or 25 nM for cysteine proteinases purified from a protozoan parasite Entamoeba histolytica or the free living nematode Caenorhabditis elegans, respectively. The 5' end of the OV7 clone was isolated by polymerase chain reaction and sequenced, thus extending the previous cDNA clone to 736 base pairs. This represents the complete coding sequence of the mature onchocystatin (130 amino acids). A hydrophobic leader sequence of 32 amino acids was found, indicating a possible extracellular function of the onchocerca cysteine proteinase inhibitor.

Immunohistological and electron microscopic studies of microfilariae in skin and lymph nodes from onchocerciasis patients after ivermectin treatment.

Microfilariae were studied in skin and lymph node biopsies from Liberian patients with generalised onchocerciasis 12-78 hours after administration of a single dose of 150 micrograms/kg body weight using histology, transmission electron microscopy and immunocytological staining with antibodies against an immunodominant antigen of Onchocerca volvulus. Most microfilariae in the skin appeared morphologically intact and beginning signs of degeneration were seen only on the ultrastructural level. The densities of microfilariae in the lymph nodes were about thousandfold higher in ivermectin treated patients. More than 90% of the microfilariae in the lymph nodes showed distinct signs of degeneration. Early changes were seen in the muscle cells. The disintegrating microfilariae in the lymph nodes were always encircled by eosinophils or macrophages or both cells. Immunohistological staining with antifilarial antibodies increased the detection of small and disintegrating pieces of microfilariae considerably.

Fine structure of microfilariae in the skin of onchocerciasis patients after exposure to amocarzine.

Transmission electron microscopy was used to demonstrate the effects of amocarzine (CGP 6140) on the fine structure of Onchocerca volvulus microfilariae (mf) in skin biopsies from patients treated orally in Guatemala or transepidermally exposed in Liberia. After 6-10 hours exposure to the drug most mf did not show any alterations and only a few mf contained increased numbers of vacuoles in the cytoplasm and clefts between cuticle and hypodermis. At 20-48 hours after treatment most of the mf showed distinct signs of damage. Most frequently seen was disintegration of the cytoplasm of the afibrillar portion of the muscle cells. Some mf showed also disintegration of the myofilaments and of the internal structure of the mitochondria in the muscle cells. Other signs were progressive separation of the cuticle from the hypodermis, increase of intracellular vacuoles and clefts and in some mf condensation of the cytoplasm. The type and the site of the morphological alterations were the same after both forms of amocarzine administration. The degree of morphological changes increased with the length of time of exposure to the drug. Microfilariae with morphological alterations were nearly always surrounded by adherent host cells, mostly eosinophils and macrophages.

Onchocerca fasciata Railliet and Henry, 1910 and its nodule development in camels in Saudi Arabia.

A total of 192 male camels of three age groups (young, adult and old) from Saudi Arabia were examined for Onchocerca fasciata infection by detection of microfilariae in skin snips and nodules in the nuchal ligaments and subcutaneous tissues of the neck and shoulder. The overall prevalence rates were 10.9 and 33.3%, respectively. The prevalence rate by the skin snip technique and the number of microfilariae per gram of skin were higher in young and adult camels than in old camels. However, the prevalence rate by the detection of nodules and the number of nodules per infected camel, increased with increase in age of the camels. An increase in size and weight of nodules was reported with an increase in age of the camels. Nodules varied in diameter from 2 to 36 mm and in weight from 0.5 to 5.0 g. The overall percentage of soft viable and calcified nodules was 42.5 and 57.5%, respectively. The viability of worms decreased, but calcification increased with increased age of the camels. Four levels of degeneration and calcification of worms were described following scanning electron microscopy.

Characterization of an Onchocerca volvulus cDNA clone encoding a genus specific antigen present in infective larvae and adult worms.

The isolation and characterization of a recombinant cDNA clone (OV7) expressing an antigen present in Onchocerca volvulus infective larvae and adult stages is described. Using chimpanzee antiserum generated against irradiated infective larvae, we isolated a cDNA clone from a lambda gt11 cDNA expression library derived from adult O. volvulus mRNA. The open reading frame encodes 131 amino acids corresponding to a 15.2-kDa protein. Affinity purified antibodies which bound specifically to OV7 fusion polypeptide recognized a single antigen with an apparent molecular weight of 17,000 in extracts of L3, L4 and adult worms. Immunoelectron microscopy established that the antigen encoded by this clone is present in the hypodermis and the basal layer of the cuticle of L3 and female adult worm, and in the egg shell around developing microfilariae. Since the OV7 fusion polypeptide is onchocerca-specific and is recognized specifically by sera from onchocerciasis patients, and sera from non-patent but infected chimpanzees, and not by sera from patients with other filarial parasites, it may have potential as an antigenic component in a test for detection of non-patent and patent infections of O. volvulus. The OV7 amino acid sequence contains residues that have a probable homology with the cysteine proteinase inhibitor superfamily.

Surface antigens of male worms and microfilariae of Onchocerca gibsoni.

Living adult males and microfilariae of the cattle filarial parasite Onchocerca gibsoni were externally labelled with radioactive iodine using the iodogen and Bolton-Hunter procedures. Characterization of labelled surface proteins by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis revealed clear cut differences in the two life cycle stages. In addition, the two radiolabelling procedures yielded some differences in the profiles of radiolabelled surface proteins for both adults and microfilariae. Immunoprecipitation analysis revealed a number of labelled antigens recognized by antibodies in human onchocerciasis serum pools, thereby demonstrating the usefulness of O. gibsoni as a model in Onchocerca volvulus vaccine studies. The reactivity of microfilarial antigens extended to antibodies from other human nematode infections, whereas male surface antigens, particularly those of low molecular weight, were Onchocerca specific. This indicates that O. gibsoni can provide a convenient source of specific diagnostic antigen.

The conditions required for the maintenance of Onchocerca lienalis microfilariae in vitro.

Microfilariae of the bovine parasite Onchocerca lienalis were maintained in vitro using a tick cell line as a feeder layer, and under the conditions provided would develop to the sausage stage. A number of media supported this achievement with CMRL 1066 producing the highest yields, particularly when used in conjunction with either medium 199, HAM's F-12, RPMI 1640 or Mark's M20. The addition of inactivated foetal calf serum (iFCS) suppressed development; on the other hand, the addition of tryptose phosphate broth (TPB) enhanced it. The pH range of the medium preferred by the developing worms was 7.35-7.85, and the most favourable osmolality lay in the range 360 to 390 mosM/kg. Insect-derived hormones did not improve yields of developed larvae nor promote moulting to the second larval stage. The culture conditions described in this work, which favour parasite survival and development, provide further insights into the physiological requirements of filariae as well as guide lines to achieving successful in vitro maintenance of Onchocerca sp. A morphological examination of the developing larvae was carried out at electron microscopical level.

Morphological demonstration of essential functional changes after in vitro and in vivo transition of infective Onchocerca volvulus to the post-infective stage.

Transmission electron microscopic investigation of the morphogenesis of Onchocerca volvulus through the third moult to the post-infective stage revealed essential alterations in various larval organs. Complete rebuilding of surface structures, the reduction of secretory granules in the glandular oesophagus, the unfolding of the intestine, an increase in the number of nerve fibres in the nerve ring and novel sensory papillae were significant findings. Transition from third- to fourth-stage larvae (L4) started as early as 48 h after transfer to vertebrate conditions in vivo in surrogate hosts and in vitro. After a resting period of about 60 h to enable a reduction in gland size and the loosening of the old cuticle and formation of the new one, the larvae started to cast the infective-stage cuticle. Young L4 exhibited a thin, monolayered cuticle and did not rebuild a glandular oesophagus. The body cavity widened, the intestine unfolded and the increased number of microvilli indicated the resumption of metabolic activity.

Morphological alterations of male Onchocerca volvulus after in vitro exposure to mel w and milbemycin a confirming the results of viability tests.

The suitability of two viability parameters used for screening of antifilarial activities of new compounds was examined by parallel observation of the morphology. Male Onchocerca volvulus were exposed in vitro to 10 mumol mel w and milbemycin a and examined by light and transmission electron microscopy. The viability was assessed by measurement of the motility, using a micromotility meter and by determination of tetrazolium reduction. Already twelve hours after exposure to mel w the muscles of the body wall showed severe damage. After 36 hours the other tissues revealed degenerative changes and after 60 hours disintegration of all tissues was observed. Effects on the morphology caused by milbemycin a were seen earliest after 60 hours. Condensed cytoplasm in the hypodermal layer and beginning degeneration of spermatogenic stages indicated drug activity. The time-point of appearance of these drug induced morphological alterations was in accordance with the decrease of the motility indices and the degree of tetrazolium reduction. Morphological alterations indicating irreversible damage of worm tissues are a reliable parameter to detect macrofilaricidal activity. The good agreement between the results of the morphological examination and the assessment of the motility and the tetrazolium reduction confirms the suitability of the latter two assays for in vitro drug screening with O. volvulus.

Onchocerca volvulus: biochemical and morphological characteristics of the surface of third- and fourth-stage larvae.

The annulated cuticles of third- and fourth-stage larvae of Onchocerca volvulus have the typical structure of other nematodes but the cuticle of fourth-stage larvae was thinner. The surface of the third-stage larva was wrinkled and fuzzy, while that of the fourth-stage was smooth. Intermediate stages in the formation of the new cuticle and epicuticle beneath the old basal layer and of the separation of the cuticles are shown. Monoclonal antibodies specific to the surface of third-stage larvae did not react with the surface of the fourth-stage larvae. Binding of the monoclonal antibodies to the third-stage larvae was abrogated by treatment of the worms with trypsin and proteinase K, but was unaffected by treatment with periodate or the detergents sodium deoxycholate and SDS. The lectins RCA120 and WGA, but not any of the other lectins tested, bound only to the surface of fourth-stage larvae, and not to that of third-stage larvae. The surfaces of third- and fourth-stage larvae were shown to be different and contained stage-specific surface epitopes.